The present invention relates to a process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid by making use of the action of a lyase possessed by a microorganism.
S,S-2-Hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid is a compound which is expected to be used in the fields of photography, detergents, paper making, etc. as a biodegradable chelating agent.
As to a process for producing 2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid (hereinafter abbreviated as HPDDS), there have been disclosed a process for producing its stereoisomer mixture (a mixture of meso-compound and racemic modification) from maleic acid and 2-hydroxypropylenediamine (alias: 1,3-diamino-2-propanol) (cf. U.S. Pat. No. 3,158,635) and a process for producing its optically active S,S-isomer from S-aspartic acid, which is an optically active substance, and 1,3-dichloro-2-propanol (cf. Zhurnal Obshuchei Khimii, Vol. 49, p. 663, 1979). Further, it is known that among the three isomers of S,S-, R,R- and meso-, only the S,S-isomer is easily biodegradable (cf. JP-A-8-507805).
On the other hand, the present inventors have previously found a novel lyase activity of a microorganism which catalytically converts fumaric acid and ethylenediamine into S,S-ethylenediamine-N,Nxe2x80x2-disuccinic acid (said lyase being hereinafter designated as ethylenediamine disuccinic acid ethylenediamine lyase and abbreviated as EDDS-ase) and proposed an economical and efficient process for producing an optically active aminopolycarboxylic acid which makes use of the above-mentioned catalytic action (cf. JP-A-9-140390). The inventors have further developed various technologies regarding the process for producing such optically active aminopolycarboxylic acids (cf., for example, JP-A-9-289895, JP-A-10-52292, JP-A-10-218846 and JP-A-10-271999). However, in almost all cases, an enzyme has a strict substrate specificity, and it has been utterly unknown whether HPDDS can be synthesized by the action of EDDS-ase or not.
The object of the present invention is to provide an economically advantageous process for producing S,S-HPDDS which does not use an expensive optically active substance.
The present inventors have made extensive study to attain the above-mentioned object and resultantly found that, by the action of EDDS-ase, S-2-hydroxypropylenediamine-N-monosuccinic acid is synthesized from one molecule of fumaric acid and one molecule of 2-hydroxypropylenediamine and further one molecule of fumaric acid reacts thereto, whereby S,S-HPDDS can be synthesized stereospecifically, and that S,S-HPDDS can be similarly synthesized from maleic acid and 2-hydroxypropylenediamine by combining the above-mentioned action of EDDS-ase with the action of maleic acid isomerase. The present invention has been accomplished on the basis of the above findings.
Thus, according to the present invention, there are provided
(1) a process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid which comprises producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid, in an aqueous medium containing fumaric acid and 2-hydroxypropylenediamine as substrates, from said substrates by the action of ethylenediamine disuccinic acid ethylenediamine lyase of a microorganism origin,
(2) a process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid which comprises producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid, in an aqueous medium containing maleic acid or maleic anhydride and 2-hydroxypropylenediamine as substrates, from said substrates by combined actions of ethylenediamine disuccinic acid ethylenediamine lyase and maleic acid isomerase each of a microorganism origin,
(3) the process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid described in (1) or (2) above wherein at least one metal ion selected from the group consisting of alkaline earth metals, iron, zinc, copper, nickel, aluminum, titanium and manganese is made to exist in the aqueous medium containing the substrates,
(4) the process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid described in (3) above wherein the metal ion is at least one metal ion selected from the group consisting of magnesium, manganese and iron,
(5) the process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid described in (1) or (2) above wherein a content of R-2-hydroxypropylenediamine-N-monosuccinic acid contained as an impurity in the aqueous medium containing the substrates is 2.5% by mole or less of the theoretical value of S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid to be formed,
(6) a process for producing an S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid alkali metal salt which comprises adding an alkali hydroxide to the reaction product mixture obtained in (3) or (4) described above to separate and recover as an insoluble precipitate the metal ions, which had been made to exist, and simultaneously to convert the S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid into its alkali metal salt,
(7) the process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid described in (3) or (4) above wherein the separated and recovered insoluble precipitate is reused as a metal ion source,
(8) the process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid described in any of (1) to (5) above which comprises adding at least one organic acid selected from the group consisting of fumaric acid, maleic acid and maleic anhydride to the reaction product mixture, recovering S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid or its salt as an insoluble substance and reusing the resulting supernatant for the reaction,
(9) the process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid described in any of (1) to (5) above which further includes the step of precipitating and recovering S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid as crystalline powder from the reaction product mixture under an acidic condition by use of a mineral acid,
(10) a crystalline powder of S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid in which the sum of contents of cyclic compounds represented by the following structural formulas [1] and [2] is 1% by mole or less relative to S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid, 
(11) the process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid described in (1) above wherein the ethylenediamine disuccinic acid ethylenediamine lyase originates from a microorganism belonging to the genus Brevundimonas, the genus Paracoccus, the genus Sphingomonas, the genus Acidovorax, the genus Pseudomonas or the genus Burkholderia or from a microorganism transformed by a gene DNA which codes ethylenediaminedisuccinic acid ethylenediamine lyase of these microorganisms origin, and
(12) the process for producing S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid described in (2) above wherein the ethylenediamine disuccinic acid ethylenediamine lyase originates from a microorganism belonging to the genus Brevundimonas, the genus Paracoccus, the genus Sphingomonas, the genus Acidovorax, the genus Pseudomonas or the genus Burkholderia or from a microorganism transformed by a gene DNA which codes ethylenediamine disuccinic acid ethylenediamine lyase of these microorganisms origin and the maleic acid isomerase originates from a microorganism belonging to the genus Alcaligenes, the genus Pseudomonas, the genus Xanthomonas or the genus Bacillusor or from a microorganism transformed by a gene DNA which codes maleic acid isomerase of these microorganisms origin.
Cultivation of Microorganism
The microorganisms used in the present invention are as described later.
The kind of culture media for the microorganism used in the present invention is not particularly limited and both a synthetic medium and a natural medium may be used so long as they appropriately contain an assimilable carbon source, nitrogen source, inorganic salt and further a slight amount of an organic nutrient. When obtaining strain cells having a high activity is desired, for example, in the case of microorganisms having an EDDS-ase activity, an amino acid such as HPDDS, ethylenediamine-N,Nxe2x80x2-disuccinic acid, ethylenediamine-N-monosuccinic acid, aspartic acid, glutamic acid and histidine or fumaric acid etc. is added to the culture medium and, in the case of microorganisms having a maleic acid isomerase activity, maleic acid and the like is added to the medium. The cultivation conditions may vary depending on the strain cells and the culture medium. The pH of the culture medium is in the range of preferably from 4 to 10, more preferably from 6 to 9. The cultivation temperature is preferably 20-40xc2x0 C., but in the case of a thermophilic microorganism such as those of the genus Bacillis, a cultivation temperature of about 50-70xc2x0 C. may be used in some cases. The cultivation is conducted until the activity reaches the maximum, if necessary with aeration and/or stirring.
Removal of Fumarase Activity of Microorganism
Removal of the fumarase activity present in microorganisms may be conducted for strain cells or a substance obtained by treating the strain cells (in the present description, refers to disrupted strain cells, strain cell extract, an extracted crude or purified enzyme, imobilized strain cells or enzyme, and strain cells or enzyme subjected to a treatment with chemicals (e.g. stabilizing treatment)) at a treating pH in the range of preferably from 8 to 10.5, more preferably from 8.5 to 10, at a treating temperature preferably in the range of from freezing temperature to 55xc2x0 C. and for a period of time not particularly limited (cf. JP Application No. 9-311046).
S,S-HPDDS Producing Reaction
A reaction for producing S,S-HPDDS is preferably conducted by bringing strain cells or a substance obtained by treating the strain cells, in an aqueous medium containing fumaric acid and 2-hydroxypropylenediamine of substrates and, according to necessity, metal compounds, which serve as the source of metal ions that participate in the reaction, and salts etc. having a buffering ability to be added for stabilizing the enzyme, into contact with the substrates, but it may also be conducted by adding fumaric acid, 2-hydroxypropylenediamine and said metal compound directly to the strain cell culture broth.
The reaction is conducted in the temperature range of preferably from 0xc2x0 C. to 60xc2x0 C., more preferably from 20xc2x0 C. to 45xc2x0 C., and in the pH range of preferably 4-11, more preferably 7-10. The concentration of fumaric acid used in the reaction is preferably 0.01-3M (mol/l) though it varies depending on the reaction temperature and pH; the presence of the acid as a precipitate due to its concentration exceeding the saturation solubility is permissible because the precipitate goes into solution with the progress of the reaction. The concentration of 2-hydroxypropylenediamine is preferably 0.01-2M.
The forming ratio of S-2-hydroxypropylenediamine-N-monosuccinic acid, which is an intermediate of the present reaction, to S,S-HPDDS can be varied according to necessity by varying the molar ratio of fumaric acid to 2-hydroxypropylenediamine. In this case, the forming ratio of S-2-hydroxypropylenediamine-N-monosuccinic acid increases when the ratio of 2-hydroxypropylenediamine to fumaric acid is increased.
The amount of the microorganism or the like to be used is preferably 0.01-5% by weight in terms of dry strain cells relative to the substrate.
Irrespective of whether the starting material concerned is 2-hydroxypropylenediamine or fumaric acid, a reaction system which can synthesize the two starting materials from other compounds can be made to coexist with the present system so long as an effect which constitutes the gist of the present invention can be obtained.
When maleic acid isomerase is made to coexist in the reaction, maleic acid or its salt can be used as a substrate in place of the above-mentioned fumaric acid. Maleic anhydride can be used similarly because it is easily converted into maleic acid in an aqueous solution. The conditions for such reactions are generally similar to those wherein fumaric acid is used as the starting material, though in some cases they vary depending on EDDS-ase which is made coexistent.
Reaction in the Presence of Metal Ion
The present reaction is an equilibrium reaction and seemingly stops in the middle thereof. Thus, the equilibrium can be shifted to the product side and the yield can be improved by making present in the reaction system a polyvalent metal ion which can be coordinated to S,S-HPDDS.
The metal ion in the present invention is not particularly limited so long as it is a metal ion which can be chelated to S,S-HPDDS to form a complex. It may be, for example, ions of simple elements of heavy metals and alkaline earth metals or ions of coordination compounds. Specific examples thereof include ions of heavy metals such as Fe(II), Fe(III), Zn(II), Cu(II), Ni(II), Co(II), Al(III), Mn(II) and Ti(IV); and ions of alkaline earth metals such as Mg(II), Ca(II) and Ba(II). In actual practice, it is preferable to add hydroxides, oxides and their salts, and compounds with sulfuric acid, hydrochloric acid, nitric acid, acetic acid and carbonic acid of these metal ions. Accordingly, even when these compounds are present in the reaction mixture in the form of ions comprising a metal or a non-metallic element or such ions are generated after the addition, they are usable so long as they can coordinate with S,S-HPDDS and can give the intended effect of the present invention. These ions or compounds may be used in a combination of two or more thereof.
Some of the metal compounds or the salts comprising the metal compound and fumaric or maleic acid have a low solubility. However, the presence of such compounds or salts over saturation, that is, in the form of suspension, is permissible because S,S-HPDDS which is formed with the progress of reaction will chelate therewith thereby to yield the intended effect of the present invention. These metal compounds may be added either in a lump at the initiation of the reaction or in the course of the reaction.
The amount of the metal compound used as the metal source and added to the reaction mixture is preferably 0.01-2 times by mole the amount of S,S-HPDDS formed.
In controlling the pH, when a metal ion is not added or when an alkaline earth metal is added, since the pH tends to decrease, the pH control can be done with an alkali such as alkali metal hydroxides, 2-hydroxypropylenediamine and alkali metal salt or ammonium salt of S,S-HPDDS, and when an ion of a metal other than alkaline earth metals is added, since the pH tends to increase, it can be done with an acid such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, fumaric acid, maleic acid and S,S-HPDDS.
When the metal complex of S,S-HPDDS is required, the intended compound can be directly obtained, after conducting the reaction in the presence of a predetermined metal ion, through operations of pH control, concentration, etc. On the other hand, for collecting S,S-HPDDS from the reaction product mixture, it can be effected by conducting an acid precipitation as described later. However, in a system wherein a complex which is stable at the pH of the acid precipitation has been formed, for example in the case that the reaction has been conducted in the presence of a heavy metal ion such as an iron ion, said metal ion needs to be removed prior to the acid precipitation. Therefore, when S,S-HPDDS is needed, it is effective to conduct the reaction in the presence of an ion of alkaline earth metals such as magnesium and calcium, which does not need the above-mentioned removal operation, at the time of acid precipitation.
The effect of the metal ion addition is thought to be attributed to the shift of the chemical equilibrium point from the substrate side to the product side caused by the metal ion. Since, in general, a chemical equilibrium point is independent on the kind of a catalyst, the chemical equilibrium point in the present invention shows a constant value for all catalysts so long as it is not affected by a side reaction or other reactions. Therefore, the effect of the metal ion addition is not particularly related to which microorganism is the origin of the EDDS-ase of the catalyst.
Suppression of Meso-HPDDS Formation During Reaction
In the reaction product mixture as it is or when an aqueous alkali metal salt solution is needed as described later, a contamination by meso-HPDDS is usually unfavorable because not only it lowers a chemical purity of intended S,S-HPDDS but also the meso-isomer is poor in biodegradability as compared with S,S-HPDDS as mentioned above.
As to the cause of meso-HPDDS contamination, the following schema is supported by experimental facts. First, racemic modification of 2-hydroxypropylenediamine-N-monosuccinic acid is formed from fumaric or maleic acid and 2-hydroxypropylenediamine without the aide of a microorganism catalyst. Then, of two optical isomers of S and R, R-hydroxypropylenediamine-N-monosuccinic acid reacts with fumaric acid through the catalytic action of EDDS-ase to form meso-HPDDS (the S-isomer is converted to S,S-HPDDS).
The above-mentioned formation of the racemic-2-hydroxypropylenediamine-N-monosuccinic acid is the more marked when the temperature is the higher, the pH is the higher, the substrate concentration is the higher and the period of time of an exposure of the substrate to these conditions is the longer. The formation is particularly marked when the substrate concentration is high; in other words, the racemic compound is mainly formed during the preparation of the aqueous substrate solution rather than during the reaction. That is, the contamination by meso-HPDDS can be markedly suppressed if the formation of the racemic-2-hydroxypropylenediamine-N-monosuccinic acid during the preparation of the aqueous substrate solution is suppressed. Desirably, the content of R-2-hydroxypropylenediamine-N-monosuccinic acid in the aqueous substrate solution is controlled to 2.5% by mole or less of the theoretical value of S,S-2-hydroxypropylenediamine-N,Nxe2x80x2-disuccinic acid to be formed.
Recovery and Recycle Use of Metal in Adding Reaction of Metal Ion, and Production of S,S-HPDDS Alkali Metal Salt
After a completion of the reaction, the strain cells or the substance obtained by treating the strain cells are removed by filtration or centrifugation, etc., then an alkali hydroxide such as sodium hydroxide and potassium hydroxide is added thereto until the metal ion becomes insolubilized, and the insolubilized precipitate is removed by a conventional means for solid-liquid separation, such as filtration or centrifugation, whereby S,S-HPDDS alkali metal salt can be obtained as a supernatant. The recovered precipitate can be reused as the metal ion source in the above-mentioned reaction.
The amount of the alkali hydroxide to be used is preferably 2-6 times by mole equivalent, more preferably 3-4.5 times by mole equivalent, in terms of the final concentration in the reaction mixture (including the amount added during the preparation of the substrate solution or during the reaction), of the amount of S,S-HPDDS contained in the reaction mixture, though it may vary depending on the kind of metal ions used in the reaction. The alkali hydroxide may be used in a mixture of two or more kinds thereof or in a combination with other alkalis.
In adding the alkali hydroxide, when it is added at once in a short time, the precipitating insolubilized product tends to become fine particles and difficult to separate; however, an addition over a sufficiently long period of time is preferable because particles with good sedimentation property are obtained thereby. Both when the alkali hydroxide is added to the reaction product mixture or, conversely, when the reaction product mixture is added to the alkali hydroxide, a solidification will take place at the instant when liquid drop added comes into contact with the other liquid, so that a uniformity in the vessel tends to be difficult to be maintained, particle diameters tend to be non-uniform, and too fine particles tend to be formed. As a more recommendable procedure for such a case, the reaction product mixture and the alkali hydroxide are fed together into a suitable crystallization vessel and, preferably, the simultaneous feeding and withdrawing of the slurry from the vessel are conducted continuously, whereby relatively large particles of improved separability can be obtained.
Acid Precipitation and Recovery of S,S-HPDDS by Use of Fumaric Acid or Maleic Acid and Recycle Use of Supernatant
The acid precipitation of S,S-HPDDS can be conducted by using fumaric acid or maleic acid (including maleic anhydride and substances obtained by treating it), which are the substrates of the reaction. The conditions for the acid precipitation are selected from ranges wherein S,S-HPDDS precipitates and these substrates are dissolved because the acid precipitation is intended for S,S-HPDDS recovery and, at the same time, for the recycle use of the supernatant in the reaction. The amount of fumaric acid or maleic acid to be added is preferably 0.2-3 times by mole, more preferably 0.8-2.4 times by mole, of the amount of S,S-HPDDS. The temperature is controlled in the range of preferably from about 0xc2x0 C. to about 80xc2x0 C., more preferably from 10xc2x0 C. to 60xc2x0 C., by if necessary gradually cooling the slurry after a part of the S,S-HPDDS has been precipitated or seed crystals of S,S-HPDDS have been added. When the reaction is conducted as a continuous process, under the conditions as described above, fumaric acid or maleic acid and the above-mentioned reaction product mixture may be fed so as to give a residence time of preferably from about 0.5 to about 10 hours, more preferably from 1 to 5 hours, and the S,S-HPDDS crystal slurry obtained can be withdrawn continuously or intermittently.
The precipitated crystals can be collected by conventional means such as filtration and centrifugation. Subsequently, salts in crude crystals formed at the time of acid precipitation and cyclized products described later are washed away by using water or an organic solvent. The method for the washing also is not particularly limited and can be conventional ones such as linsing and slurry washing.
Wet crystals obtained after washing is dried preferably at a temperature so as to keep a product temperature of not higher than 80xc2x0 C.
Supernatant obtained after the recovery of S,S-HPDDS can be reused for the above-mentioned reaction after having been mixed with a specified amount of 2-hydroxypropylenediamine and an acid, alkali, etc. for pH control.
Acid Precipitation and Recovery of S,S-HPDDS by Use of Mineral Acid
For the crystallization, the above-mentioned reaction product mixture or an aqueous S,S-HPDDS alkali metal salt solution or a concentrated product thereof etc. are, in the case of conducting in a batch process, conditioned to a pH range of preferably from about 1.8 to about 4.5, more preferably from 2.0 to 4.0 by using a mineral acid such as sulfuric acid and hydrochloric acid and to a temperature range of preferably from about 40xc2x0 C. to about 80xc2x0 C., more preferably from 40xc2x0 C. to 60xc2x0 C., and, if necessary a part of the S,S-HPDDS has been precipitated or seed crystals of S,S-HPDDS have been added, and then it is gradually cooled. The cooling temperature is preferably not higher than about 40xc2x0 C., more preferably 30xc2x0 C. to 0xc2x0 C. When the crystallization is conducted in a continuous process, the initial charge of the above-mentioned reaction product mixture, etc. is conditioned to a pH range of preferably from about 1.8 to about 4.5, more preferably from 2.0 to 4.0 and a temperature range of preferably from 0xc2x0 C. to 40xc2x0 C., then the mineral acid and the above-mentioned reaction product mixture and an aqueous S,S-HPDDS alkali metal salt solution or a concentrated product thereof etc. are fed so as to give a residence time of preferably from about 0.5 to about 10 hours, more preferably from 1 to 5 hours, though the residence time may vary depending on the conditions of pH and temperature, and the resulting S,S-HPDDS slurry is withdrawn continuously or intermittently.
Though the pH increases along with the precipitation of the crystals, pH may be adjusted, according to necessity, to a predetermined value by use of a mineral acid such as sulfuric acid and hydrochloric acid. When the crystallization pH is selected to about 4 or above, S,S-HPDDS tends to precipitate as salt of cations contained in the reaction mixture. Since such salts have sometimes their own merits that, for example, a mono-sodium salt of S,S-HPDDS has more affinity for water than S,S-HPDDS itself, the pH may be changed according to necessity to obtain an intended salt.
On the other hand, cyclized products originated from HPDDS represented by the following structural formulas [1] and [2] tend to be formed the more easily as the pH is the lower and the temperature is the higher and the time of exposure to these conditions is the longer. The formation of the cyclized products results in a decrease of a recovery rate of S,S-HPDDS crystals. Moreover, the cyclized products remaining in the supernatant adhere to the crystals to result in a cause for a lowering of the quality of the intended product. An adoption of the conditions described above as preferable can keep the formation of the cyclized product at a less extent. According to the present acid precipitation method, the crystals of S,S-HPDDS can be ultimately obtained in a yield of 90% or more.
Collection and drying etc. of the precipitated crystals can be conducted in the same manner as described for the above-mentioned acid precipitation using fumaric acid and maleic acid. 
Microorganism Having EDDE-ase
Examples of the microorganism having an EDDS-ase activity include microorganisms belonging to the genus Burkholderia, the genus Acidovorax, the genus Pseudomonas, the genus Paracoccus, the genus Sphingomonas and the genus Brevundimonas, and further transformants obtained by introducing a gene which codes EDDS-ase into a microorganisms belonging to the genus Esherichia or the genus Rhodococcus used as a host.
Specific examples include Burkholderia sp. KK-5 (FERM BP-5412), Burkholderia sp. KK-9 (FERM BP-5413), Acidovorax sp. TN-51 (FERM BP-5416), Pseudomonas sp. TN-131 (FERM BP-5418), Paracoccus sp. KK-6 (FERM BP-5415), Paracoccus sp. TNO-5 (FERM BP-6547), sphingomonas sp. TN-28 (FERM BP-5419), Brevundimonas sp. TN-30 (FERM BP-5417) and Brevundimonas sp. TN-3 (FERM BP-5886) and further transformants obtained by using Escherichia coli JM109 [Escherichia coli ATCC 53323] or Rhodococcus rhodochrous ATCC 17895 as a host.
Among the above-mentioned microorganisms, the strains KK-5, KK-9, TN-51, TN-131, KK-6, TN-28, TN-30 and TN-3 were newly isolated from the natural world by the present inventors and have been deposited with National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (Higashi 1-1-3, Tsukuba-shi, Ibaraki-ken, Japan (postal code number 305-8566)) under the above-mentioned accession numbers. The mycological properties of these strains are also described in the above-mentioned JP-A-9-140390 and JP-A-10-52292.
The strain TNO-5 was also newly isolated from the natural world by the present inventors and has been deposited with the above-mentioned National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry under the above-mentioned accession number. Bachteriological properties thereof are as follows.
As a result of classification based on the above-mentioned bacteriological properties according to the description given in Bergey""s Manual of Systematic Bacteriology, Vol. 9 (1990), the strain TNO-5 was identified as a bacterium belonging to the genus Paracoccus. Incidentally, it has been confirmed that the strain TN-3 belongs to the diminuta sp.
The strain Esherichia coli JM109 (Esherichia coli ATCC53323 strain) and the strain Rhodococcus rhodochrous ATCC17895 are known and are easily available from the American Type Culture Collection (ATCC). Transformants obtained by using these strains as a host and introducing thereinto plasmids pEDS020 and pSE001 containing gene DNA which codes a protein having the EDDS-ase activity of the strain TN-3 have been deposited as E. coli JM109/pEDS020 (FERM BP-6161) and Rhodococcus rhodochrous ATCC17895/pSE001 (FERM EP-6548) respectively with National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
Methods for preparation of these transformants are described in JP Application No. 9-60077 filed by the present applicant.
Microorganism Having Maleic Acid Isomerase
The microorganism having maleic acid isomerase activity is not particularly limited so long as it is a microorganism capable of iomerizing maleic acid to fumaric acid and may be, for example, microorganisms belonging to the genus Alcaligenes, the genus Pseudomonas, the genus Xanthomonas and the genus Bacillus, and further, transformants obtained by introducing a gene which codes maleic acid isomerase originated from these microorganisms.
Specific examples of the strains include Alcaligenes faecalis sp. IFO 12669, Alcaligenes faecalis sp. IFO 13111, Alcaligenes faecalis sp. IAM1473, Alcaligenes eutrophas sp. IAM12305, Pseudomonas fluolescens sp. ATCC 23728, Xanthomonas maltophilia sp. ATCC13270, Bacillus sp. MI105 (FERM BP-5164), Bacillus stearothermophirus sp. MI101 (FERM BP-5160) Bacillus stearothermophirus sp. MI102 (FERM BP-5161), Bacillus brevis sp. MI103 (FERM BP-5162) and Bacillus brevis sp. MI104 (FERM BP-5163).
These microorganisms are easily available from Institute for Fermentation, Osaka (IFO) (a foundation) (Japan); Center for Cellular and Molecular Research IAM Culture Collection, Inst. of Molecular and Cellular Biosciences (Center for Bio-information), University of Tokyo (Japan); the American Type Culture Collection (ATCC) (U.S.A); and National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (Japan).
Further, among the microorganisms having EDDS-ase, there may also be used microorganisms which have an ability to convert maleic acid into fumaric acid, that is, converts maleic acid and 2-hydroxypropylenediamine directly into S,S-HPDDS. Typical examples of such microorganisms are the above-mentioned Burkholderia sp. KK-5 (FERM BP-5412), Burkholderia sp. KK-9 (FERM BP-5413), Pseudomonas sp. TN-131 (FERM BP-5418), Paracoccus sp. KK-6 (FERM BP-5415), Sphingomonas sp. TN-28 (FERM BP-5419) and Brevundimonas sp. TN-30 (FERM BP-5417). Though microorganisms usable in the present process exist also in the natural world as described above, there may also be used transformants obtained by simultaneously introducing thereinto the maleic acid isomerase gene and the EDDS-ase gene.
The present invention is described in detail below with reference to Examples.